A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens.

A reliable extraction method is described for the preparation of total nucleic acids from at least ten plant genera for subsequent detection of plant pathogens by PCR-based techniques. The method combined a modified CTAB (cetyltrimethylammonium bromide) extraction protocol with a semi-automatic homogenizer (FastPrep) instrument) for rapid sample processing and low potential of cross contamination. The method was applied to sample preparation for PCR-based detection of 28 different RNA and DNA viruses, six viroids, two phytoplasmas and two bacterial pathogens from a range of infected host plants including sweet potato, small fruits and fruit trees. The procedure is cost-effective and the qualities of the nucleic acid preparations are comparable to those prepared by commonly used commercial kits. The efficiency of the procedure permits processing of numerous samples and the use of a single nucleic acid preparation for testing both RNA and DNA genomes by PCR, making this an appealing method for testing multiple pathogens in certification and quarantine programs.

[1]  N. Boonham,et al.  Advances in molecular phytodiagnostics – new solutions for old problems , 2006, European journal of plant pathology.

[2]  R. Jorgensen,et al.  Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Flores-Vergara,et al.  A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide , 2006, Nature Protocols.

[4]  J. Valkonen,et al.  Potyvirus Complexes in Sweetpotato: Occurrence in Australia, Serological and Molecular Resolution, and Analysis of the Sweet potato virus 2 (SPV2) Component. , 2006, Plant disease.

[5]  M. Wirthensohn,et al.  Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis). , 2003, Journal of virological methods.

[6]  J. Nap,et al.  Isolation of High Quality DNA and RNA from Leaves of the Carnivorous Plant Drosera rotundifolia , 1999, Plant Molecular Biology Reporter.

[7]  S. Saumtally,et al.  Sugarcane yellow leaf virus and sugarcane yellows phytoplasma: elimination by tissue culture , 2002 .

[8]  M. Royer,et al.  Genetic diversity in the coat protein coding region of eighty-six sugarcane mosaic virus isolates from eight countries, particularly from Cameroon and Congo , 2003, Archives of Virology.

[9]  N. Boonham,et al.  The use of real-time RT-PCR (TaqMan) and post-ELISA virus release for the detection of Beet necrotic yellow vein virus types containing RNA 5 and its comparison with conventional RT-PCR. , 2005, Journal of virological methods.

[10]  H. Gross,et al.  Two rapid microscale procedures for isolation of total RNA from leaves rich in polyphenols and polysaccharides: application for sensitive detection of grapevine viroids. , 1995, Journal of virological methods.

[11]  J. Marcos,et al.  Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents , 1996, European Journal of Plant Pathology.

[12]  J M Henson,et al.  The polymerase chain reaction and plant disease diagnosis. , 1993, Annual review of phytopathology.

[13]  N. Boonham,et al.  Development of real-time and conventional RT-PCR assays for the detection of potato yellow vein virus (PYVV). , 2006, Journal of virological methods.

[14]  T Demeke,et al.  The effects of plant polysaccharides and buffer additives on PCR. , 1992, BioTechniques.

[15]  J. Cairney,et al.  A simple and efficient method for isolating RNA from pine trees , 1993, Plant Molecular Biology Reporter.

[16]  Evaluation of reverse transcription – polymerase chain reaction assays for detecting Apple chlorotic leaf spot virus in certification and quarantine programs , 2006 .

[17]  R. Hammond,et al.  Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms , 1993 .

[18]  W. Menzel,et al.  Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control. , 2002, Journal of virological methods.

[19]  Zhihong Zhang,et al.  Detection of Strawberry RNA and DNA Viruses by RT‐PCR Using Total Nucleic Acid as a Template , 2007 .

[20]  B. Lockhart,et al.  A New Badnavirus in Ribes Species, its Detection by PCR, and its Close Association with Gooseberry Vein Banding Disease. , 2001, Plant disease.

[21]  Edwin L. Civerolo,et al.  Genome-based PCR Primers for Specific and Sensitive Detection and Quantification of Xylella fastidiosa , 2006, European Journal of Plant Pathology.

[22]  M. Schell,et al.  Cloning of the egl gene of Pseudomonas solanacearum and analysis of its role in phytopathogenicity , 1988, Journal of bacteriology.

[23]  Kevin P. Smith,et al.  Inexpensive, high throughput microplate format for plant nucleic acid extraction : Suitable for multiplex southern analyses of transgenes , 2005 .

[24]  D. Puchooa.  A simple, rapid and efficient method for the extraction of genomic DNA from lychee (Litchi chinensis Sonn.) , 2004 .

[25]  M. Halonen,et al.  Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit , 2001, Molecular biotechnology.

[26]  Wenbin Li,et al.  Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. , 2006, Journal of microbiological methods.

[27]  Ruhui Li,et al.  Detection of Geminiviruses in Sweetpotato by Polymerase Chain Reaction. , 2004, Plant disease.

[28]  K. Druffel,et al.  Occurrence of Two Little Cherry Viruses in Sweet Cherry in Washington State. , 2008, Plant disease.

[29]  Tao Wang,et al.  Isolation of RNA of high quality and yield from Ginkgo biloba leaves , 2005, Biotechnology Letters.

[30]  S. Stewart,et al.  Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts. , 2000, Journal of virological methods.

[31]  T. Candresse,et al.  A polymerase chain reaction assay adapted to plum pox potyvirus detection. , 1991, Journal of virological methods.

[32]  E. Pahlich,et al.  A rapid DNA isolation procedure for small quantities of fresh leaf tissue , 1980 .

[33]  P. Lotrakul,et al.  Cloning of a DNA-A-like genomic component of sweet potato leaf curl virus: nucleotide sequence and phylogenetic relationships. , 1999 .

[34]  Xuchu Wang,et al.  Development of an Efficient Protocol of RNA Isolation from Recalcitrant Tree Tissues , 2008, Molecular biotechnology.

[35]  Maja Ravnikar,et al.  Real‐time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics , 2007 .

[36]  Judith K. Brown,et al.  Detection of subgroup III geminivirus isolates in leaf extracts by degenerate primers and polymerase chain reaction , 1996 .

[37]  Ruhui Li,et al.  An improved reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of two cherry flexiviruses in Prunus spp. , 2005, Journal of virological methods.

[38]  A. Hayward,et al.  A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum) , 1997 .

[39]  A. Kelman.  The relationship of pathogenicity of Pseudomonas solanacearum to colony appearance in a tetrazolium medium. , 1954 .

[40]  A. Jones,et al.  Improved PCR Detection of Blackcurrant reversion virus in Ribes and Further Evidence that It Is the Causal Agent of Reversion Disease. , 2002, Plant Disease.

[41]  Sun-Hyung Kim,et al.  Rapid and Reliable Method of Extracting DNA and RNA from Sweetpotato, Ipomoea batatas (L). Lam , 2005, Biotechnology Letters.

[42]  C. Malmstrom,et al.  Multiplexed RT-PCR for streamlined detection and separation of barley and cereal yellow dwarf viruses. , 2004, Journal of virological methods.

[43]  N. Boonham,et al.  A comparison of molecular methods for the routine detection of viroids , 2000 .

[44]  John S. Hu,et al.  Use of polymerase chain reaction (PCR) to study transmission of banana bunchy top virus by the banana aphid (Pentalonia nigronervosa) , 1996 .

[45]  H. Katinger,et al.  A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees. , 1991, Journal of virological methods.